Il-2rbeta-selective agonists in combination with an anti-ctla-4 antibody or an anti-pd-1 antibody

ABSTRACT

The invention relates to (among other things) method of administering to a patient suffering from a cancer, the method comprising the steps of: (a) an IL-2Rβ-activating amount of a long acting, IL-2Rβ-selective agonist; and (b) a CTLA-4 pathway-inhibiting amount of an anti-CTLA-4 antibody or a PD-1 pathway-inhibiting amount of an anti-PD-1 antibody.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to Indian PatentApplication No. 3087/DEL/2014, filed Oct. 29, 2014, and to Indian PatentApplication No. 499/DEL/2014, filed Feb. 21, 2014, the disclosures ofwhich are incorporated herein by reference in their entireties.

FIELD

This invention relates to (among other things) the field of cancerchemotherapy and involves the treatment of an individual suffering froma cancer by administering to the patient a long acting IL-2Rαβ-selectiveagonist in combination with another pharmacologically active agent.

BACKGROUND

The interleukin-2 receptor (IL-2R) is a heterotrimeric protein expressedon the surface of certain immune cells, such as lymphocytes, that bindsand responds to the IL-2 cytokine. The IL-2 receptor is made up of 3subunits—IL-2Rα, IL-2Rβ, and IL-2Rγ, with each of IL-2Rα and IL-2Rβhaving binding affinity for IL-2 while IL-2Rγ alone has no appreciableaffinity. Thèze et al. (1994) Immunol. Today 17(10):481-486. Further,the IL-2Rαβ heterodimer has a faster association rate and a slowerdissociation rate when binding IL-2 versus either chain alone. Liparotoet al. J. Mol. Recognit. 12(5):316-321.

CD4+ regulatory T-cells, which are responsible for suppressing theimmune response, preferentially express the IL-2Rαβ form of the IL-2R.Thus, administration of compounds that are agonists for IL-2Rαβ can beexpected to suppress the immune response.

CD8+ memory T-cells, which are responsible for enhancing the immuneresponse, preferentially express the IL-2Rβ form of the IL-2R. Thus,administration of compounds that are agonists for IL-2Rβ can be expectedto enhance the immune response (by, e.g., increasing the proliferationof CD8+ memory T-cells).

Thus, administration of IL-2Rβ-selective agonists would be beneficial topatients suffering from certain cancers as doing so is expected toreduce the immune-suppressing effects of regulatory T-cells whileincreasing CD8+ memory T-cells, thereby recruiting the patient's ownimmune system to eliminate cancer cells. Optimally, such anIL-2Rβ-selective agonist would also exhibit relatively long exposurefollowing administration, thereby further improving the patient'sresponse to the treatment.

Recruiting the immune system of the cancer patient in the treatment ofcancer via administration of IL-2β-selective agonists—which is directlyimmunoactivating—can be further enhanced through the administration ofantagonists of immunosuppressive pathways (e.g., antagonists of CTLA-4and PD-1).

Thus, the present invention seeks to address (among other things) thecontinuing need to provide more effective treatments of cancers by, forexample, administering to a patient suffering from cancer anIL-2Bβ-selective agonist in combination with a pharmacological-basedantagonist of a immunosuppressive pathway.

This and other needs in the art are addressed by the present invention.

SUMMARY

In one or more embodiments of the invention, a method is provided, themethod comprising the steps of administering to a cancer patient: (a) anIL-2Rβ-activating amount of a long acting, IL-2Rβ-selective agonist; and(b) a CTLA-4 pathway-inhibiting amount of an anti-CTLA-4 antibody or aPD-1 pathway-inhibiting amount of an anti-PD-1 antibody. By way ofclarity, with regard to the sequence of steps in accordance with thismethod, unless otherwise indicated, the method is not limited to thesequence of steps and step (a) can be performed before, after orsimultaneously with, performing step (b).

Additional embodiments of the invention are set forth in the followingdescription and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1, 2 and 3 are plots of mean tumor volumes, relative tumor volumesand body weights, respectively, associated with an efficacy study of areceptor-selective, long acting IL-2 agonist in combination with ananti-CTLA-4 antibody on a CT26 tumor model, which study is furtherdescribed in Example 2.

FIGS. 4, 5 and 6 are plots of mean tumor volumes for 30 days, mean tumorvolumes for 106 days, and body weights, respectively, associated with anefficacy study of a receptor-selective, long acting IL-2 agonist incombination with an anti-CTLA-4 antibody on an EMT6 tumor model, whichstudy is further described in Example 3.

FIGS. 7 and 8 are plots of tumor volumes and body weights, respectively,associated with an efficacy study of a receptor-selective, long actingIL-2 agonist in combination with an anti-CTLA-4 antibody compared tothat of Proleukin with an anti-CTLA-4 antibody on an EMT6 tumor model,which study is further described in Example 4.

FIGS. 9 and 10 are plots of tumor volumes and body weights,respectively, for 11 days associated with an efficacy study involvingflow cytometry analysis of a receptor-selective, long acting IL-2agonist in combination with an anti-CTLA-4 antibody on an EMT6 tumormodel, which study is further described in Example 5.

FIGS. 11 and 12 each include graphs of tumor and spleen, respectively,of immune cell populations corresponding to the flow cytometry analysisfurther, described in Example 5.

FIGS. 13 and 14 are plots of mean tumor volumes and body weights,respectively, associated with an efficacy study of a receptor-selective,long acting IL-2 agonist in combination with an anti-PD-1 antibody on aCT26 tumor model, which study is further described in Example 6.

FIG. 15 shows two plots of mean tumor volumes and body weights followingPhase I of a three phase re-challenge study further described in Example7.

FIG. 16 shows two plots of relative mean tumor volumes and body weightchanges following Phase I of a three phase re-challenge study furtherdescribed in Example 7.

FIG. 17 shows a plot of relative body weight changes following Phase IIof a three phase re-challenge study further described in Example 7.

FIG. 18 shows two plots of mean tumor volumes and body weights followingPhase II of a three phase re-challenge study further described inExample 7.

FIG. 19 shows a plot of mean tumor volumes following Phase III of athree phase re-challenge study further described in Example 7.

FIG. 20 shows two plots of body weight changes and relative body weightchanges Phase III of a three phase re-challenge study further describedin Example 7.

FIG. 21 shows two plots of mean tumor volumes and individual tumorvolumes over the course of a three phrase re-challenge study furtherdescribed in Example 7.

FIG. 22 shows two plots of mean tumor volumes and body weights from day0 to day 11 in connection with a RSLAIL-2 and anti-CTLA-4 combinationstudy further described in Example 8.

FIG. 23 shows two plots of relative mean tumor volumes and body weightsfrom day 0 to day 11 in connection with a RSLAIL-2 and anti-CTLA-4combination study further described in Example 8.

FIG. 24 shows two plots of mean tumor volumes and body weights from day0 to day 11 in connection with a RSLAIL-2 and anti-PD-1 combinationstudy further described in Example 8.

FIG. 25 shows two plots of relative mean tumor volumes and body weightsfrom day 0 to clay 11 in connection with a RSLAIL-2 and anti-PD-1combination study further described in Example 8.

DETAILED DESCRIPTION

As used in this specification, the singular forms “a,” “an,” and “the”include plural referents unless the context clearly dictates otherwise.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions describedbelow.

“Water soluble, non-peptidic polymer” refers to a polymer that is atleast 35% (by weight) soluble, preferably greater than 70% (by weight),and more preferably greater than 95% (by weight) soluble, in water atroom temperature. Typically, an unfiltered aqueous preparation of a“water-soluble” polymer transmits at least 75%, more preferably at least95%, of the amount of light transmitted by the same solution afterfiltering. It is most preferred, however, that the water-soluble polymeris at least 95% (by weight) soluble in water or completely soluble inwater. With respect to being “non-peptidic,” a polymer is non-peptidicwhen it has less than 35% (by weight) of amino acid residues.

The terms “monomer;” “monomeric subunit” and “monomeric unit” are usedinterchangeably herein and refer to one of the basic structural units ofa polymer. In the case of a homo-polymer, a single repeating structuralunit forms the polymer. In the case of a co-polymer, two or morestructural units are repeated—either in a pattern or randomly—to formthe polymer. Preferred polymers used in connection with the presentinvention are homo-polymers. The water-soluble, non-peptidic polymercomprises one or more monomers serially attached to form a chain ofmonomers.

“PEG” or “polyethylene glycol,” as used herein, is meant to encompassany water-soluble poly(ethylene oxide). Unless otherwise indicated, a“PEG polymer” or a polyethylene glycol is one in which substantially all(preferably all) monomeric subunits are ethylene oxide subunits, though,the polymer may contain distinct end capping moieties or functionalgroups, e.g., for conjugation. PEG polymers for use in the presentinvention will comprise one of the two following structures:“—(CH₂CH₂O)_(n)—” or “—(CH₂CH₂O)_(n-1)CH₂C₂—,” depending upon whether ornot the terminal oxygen(s) has been displaced, e.g., during a synthetictransformation. As stated above, for the PEG polymers, the variable (n)ranges from about 3 to 4000, and the terminal groups and architecture ofthe overall PEG can vary.

“Branched,”, in reference to the geometry or overall structure of apolymer, refers to a polymer having two or more polymer “arms” extendingfrom a branch point.

A “physiologically cleavable” or “hydrolyzable” or “degradable” bond isa relatively labile bond that reacts with water (i.e., is hydrolyzed)under physiological conditions. The tendency of a bond to hydrolyze inwater may depend not only on the general type of linkage connecting twoatoms within a given molecule but also on the substituents attached tothese atoms. Appropriate hydrolytically unstable or weak linkagesinclude but are not limited to carboxylate ester, phosphate ester,anhydrides, acetals, ketals, acyloxyalkyl ether, imines, orthoesters,peptides, oligonucleotides, thioesters, and carbonates.

An “enzymatically degradable linkage” means a linkage that is subject todegradation by one or more enzymes.

A “stable” linkage or bond refers to a chemical bond that issubstantially stable in water, that is to say, does not undergohydrolysis under physiological conditions to any appreciable extent overan extended period of time. Examples of hydrolytically stable linkagesinclude but are not limited to the following: carbon-carbon bonds (e.g.,in aliphatic chains), ethers, amides, urethanes, amines, and the like.Generally, a stable linkage is one that exhibits a rate of hydrolysis ofless than about 1-2% per day under physiological conditions. Hydrolysisrates of representative chemical bonds can be found in most standardchemistry textbooks.

“Substantially”, or “essentially” means nearly totally or completely,for instance, 95% or greater, more preferably 97% or greater, still morepreferably 98% or greater, even more preferably 99% or greater, yetstill more preferably 99.9% or greater, with 99.99% or greater beingmost preferred of some given quantity.

“Pharmaceutically acceptable excipient” or “pharmaceutically acceptablecarrier” refers to a component that may be included in the compositionsof the invention causes no significant adverse toxicological effects toa patient.

The term “patient,” refers to a living organism suffering from or proneto a condition that can be prevented or treated by administration of acompound of the invention as described herein, and includes both humansand animals.

As indicated above, the present invention is directed to (among otherthings) a method of administering to a patient suffering from a cancer,the method comprising the steps of: (a) an IL-2Rβ-activating amount of along acting, IL-2β-selective agonist; and (b) a CTLA-4pathway-inhibiting amount of an anti-CTLA-4 antibody or a PD-1pathway-inhibiting amount of an anti-PD-1 antibody. With respect toadministering steps (a) and (b), these administering steps can beperformed in either order (as well as simultaneously) and the inventionis not limited in this regard. In one or more embodiments of theinvention, administering step (a) will be carried out beforeadministering step (b). In one or more embodiments of the invention,administering step (b) will be carried out before administering step(a). In one or more embodiments, both administering steps (a) and (b)will be carried out simultaneously. Further, in one or more embodiments,steps (a) and/or (b) will be administered repeatedly. In addition, in ormore embodiments, steps (a) and (b) will be carried out only once.

The treatment method described herein can continue for as long as theclinician overseeing the patient's care deems the treatment method iseffective. Non-limiting parameters that indicate the treatment method iseffective include the following: tumor shrinkage (in terms of weightand/or volume); a decrease in the number of individual tumor colonies;tumor elimination; and progression-free survival.

Exemplary lengths of time associated with the course of therapy inaccordance with the claimed method include: about one week; two weeks;about three weeks; about four weeks; about five weeks; about six weeks;about seven weeks; about eight weeks; about nine weeks; about ten weeks;about eleven weeks; about twelve weeks; about thirteen weeks;

about fourteen weeks; about fifteen weeks; about sixteen weeks; aboutseventeen weeks; about eighteen weeks; about nineteen weeks; abouttwenty weeks; about twenty-one weeks; about twenty-two weeks; abouttwenty-three weeks; about twenty four weeks; about seven months; abouteight months; about nine months; about ten months; about eleven months;about twelve months; about thirteen months; about fourteen months; aboutfifteen months; about sixteen months; about seventeen months; abouteighteen months; about nineteen months; about twenty months; abouttwenty one months; about twenty-two months; about twenty-three months;about twenty-four months; about thirty months; about three years; aboutfour years and about five years.

With regard to the frequency of administering the long acting,IL-2Rβ-agonist, one of ordinary skill in the art will be able todetermine an appropriate frequency. For example, a clinician can decideto administer the long acting, IL-2β-selective agonist relativelyinfrequently (e.g., once every two weeks) and progressively shorten theperiod between dosings as tolerated by the patient. With regard tofrequency of administering the anti-CTLA-4 antibody and anti-PD-1antibody, the frequency for these agents can be determined in a similarfashion. In addition, as some long acting, IL-2Rβ-selective agonists,anti-CTLA-4 antibodies, anti-PD-1 antibody are either in advancedclinical testing or commercially available, it is also possible to referto the literature to obtain an appropriate frequency of administration(keeping in mind that some adjustment may be necessary in view of thecombined effects of the treatment regimen).

The method described herein involves the administration of long acting,IL-2Rβ-selective agonist. In this regard, the invention is not limitedto any specific long acting, IL-2Rβ-selective agonist so long as theagonist exhibits an in vitro binding affinity for IL-2Rβ that is atleast 5 times greater (more preferably at least 10 times greater) thanthe binding affinity for IL-2Rαβ in the same in vitro model, and has atleast an effective 10-fold in vivo half-life greater than IL-2(half-life based on the in-vivo disappearance IL-2). By way of example,it is possible to measure binding affinities against IL-2 as a standard.In this regard, the RSLAIL-2 referenced in Example 1 of the presentdisclosure exhibits about a 60-fold decrease in affinity to IL-2Rαβrelative to IL-2, but only about a 5-fold decrease in affinity IL-2Rβrelative to IL-2.

Non-limiting examples of long acting, IL-2Rβ-selective agonists aredescribed in WO 2012/065086. An exemplary long acting, IL-2Rβ-selectiveagonist is RSLAIL-2 referenced in Example 1 in the present application.In this regard, RSLAIL-2 is a composition comprising compoundsencompassed by the following formula:

wherein IL-2 is a residue of IL-2, and pharmaceutically acceptable saltsthereof. In one or more embodiments, the composition contains no morethan 10% (based on a molar amount), preferably no more than 5% (based ona molar amount), of compounds encompassed by the following formula

wherein IL-2 is a residue of IL-2, (n) is an integer selected from thegroup consisting of 1, 2, 7 and >7, and pharmaceutically acceptablesalts thereof.

The method described herein involves the administration of ananti-CTLA-4 antibody or an anti-PD-1 antibody. With regard toanti-CTLA-4 antibodies, these are known and include tremelimumab andipilimumab. With regard to anti-PD-1 antibodies, these are known andinclude nivolumab and lambrolizumab, AMP-224, MDPL3280A, MEDI4736 andMSB0010718C.

Assays for determining whether a given compound can act as ananti-CTLA-4 antibody or anti-PD-1 antibody can be determined throughrouting experimentation by one of ordinary skill in the art.

In accordance with the method described herein, the long acting,IL-2Rβ-selective agonist is administered to a patient in anIL-2Rβ-activating amount. One of ordinary skill in the art can determinehow much a given long acting, IL-2Rβ-selective agonist sufficient toprovide clinically relevant agonistic activity at IL-2Rβ. For example,one of ordinary skill in the art can refer to the literature and/oradminister a series of increasing amounts the long acting,IL-2Rβ-selective agonist and determine which amount or amounts provideclinically agonistic activity of IL-2β.

In one or more instances, however, the IL-2Rβ-activating amount is anamount encompassed by one or more of the following ranges: from about0.01 to 1 mg/kg; from about 0.01 mg/kg to about 0.1 mg/kg; from about 1mg/kg to about 1000 mg/kg; from about 2 mg/kg to about 900 mg/kg; fromabout 3 mg/kg to about 800 mg/kg; from about 4 mg/kg to about 700 mg/kg;from about 5 mg/kg to about 600 mg/kg; from about 6 mg/kg to about 550mg/kg; from about 7 mg/kg to about 500 mg/kg; from about 8 mg/kg toabout 450 mg/kg; from about 9 mg/kg to about 400 mg/kg; from about 5mg/kg to about 200 mg/kg; from about 2 mg/kg to about 150 mg/kg; fromabout 5 mg/kg to about 100 mg/kg; from about 10 mg/kg to about 100mg/kg; and from about 10 mg/kg to about 60 mg/kg.

In accordance with the method described herein, a CTLA-4pathway-inhibiting amount of an anti-CTLA-4 antibody is administered ora PD-1 pathway-inhibiting amount of an anti-PD-1 antibody isadministered. One of ordinary skill in the art can determine how much agiven anti-CTLA-4 antibody or anti-PD-1 antibody is sufficient toprovide clinically relevant inhibition of the CTLA-4 pathway or PD-1pathway, respectively. For example, one of ordinary skill in the art canrefer to the literature and/or administer a series of increasing amountsthe anti-CTLA-4 antibody or anti-PD-1 antibody and determine whichamount or amounts provide clinically relevant inhibition the CTLA-4pathway or PD-1 pathway.

In one or more instances, however, the CTLA-4 and PD-1pathway-inhibiting amounts are encompassed by one or more of thefollowing ranges: from about 1 mg/kg to about 1000 mg/kg; from about 2mg/kg to about 900 mg/kg; from about 3 mg/kg to about 800 mg/kg; fromabout 4 mg/kg to about 700 mg/kg; from about 5 mg/kg to about 600 mg/kg;from about 6 mg/kg to about 550 mg/kg; from about 7 mg/kg to about 500mg/kg; from about 8 mg/kg to about 450 mg/kg; from about 9 mg/kg toabout 400 mg/kg; from about 5 mg/kg to about 200 mg/kg; from about 2mg/kg to about 150 mg/kg; from about 5 mg/kg to about 100 mg/kg; fromabout 10 mg/kg to about 100 mg/kg; and from about 10 mg/kg to about 60mg/kg.

For confirmation, as used herein with regard to CTLA-4 and PD-1pathway-inhibiting amounts of the of the anti-CTLA-4 antibody oranti-PD-1 antibody, respectively, the, amount and extent of theinhibition can vary widely and the combination of either of these withthe long acting, IL-2Rβ-selective agonist can still be effective. Forexample, an amount of the anti-CTLA-4 antibody or anti-PD-1 antibodythat only minimally inhibits the CTLA-4 or PD-1 pathways, respectively,can still be an inhibiting amount as used herein so long as the methodof the claimed invention results in a clinically meaningful response. Sotoo, an amount of a long acting, IL-2Rβ-selective agonist that exhibitsonly minimal agonist activity IL-2Rβ for a sufficiently extended periodof time can still be a long acting, IL-2Rβ-selective agonist so long asthe method of the claimed invention results in a clinically meaningfulresponse. In some instances, due to (for example) synergistic responses,minimal inhibition of the CTLA-4 or PD-1 pathways may only be requiredin the presence of the long acting, IL-2Rβ-selective agonist. In stillother instances, due to (for example) synergistic responses, minimalagonist activity of IL-2Rβ may be required in the presence of CTLA-4 andPD-1 pathway inhibition.

The actual dose to be administered will vary depend upon the age,weight, and general condition of the subject as well as the severity ofthe condition being treated, the judgment of the health careprofessional, and conjugate being administered.

The invention provides a method for that is useful for (among otherthings) treating a patient suffering from a condition that is responsiveto treatment with the compound. For example, patients may be responsiveto the individual agents alone as well as: the' combination, but aremore responsive to the combination. By way of further example, patientsmay be non-responsive to one of the individual agents, but areresponsive to the combination. By way of still further example, patientsmay be non-responsive to either of the individual agents alone, but areresponsive to the combination.

The method comprises administering a therapeutically effective amount ofactive agents via injection. Other modes of administration are alsocontemplated, such as pulmonary, nasal, buccal, rectal, sublingual andtransdermal. As used herein, the term “parenteral” includessubcutaneous, intravenous, intra-arterial, intraperitoneal,intracardiac, intrathecal, and intramuscular injection, as well asinfusion injections. Each pharmacological component of the method can beadministered separately. Alternatively, if administration of twopharmacological components is desired to be simultaneous—and the twopharmacological components are compatible together and in a givenformulation—then the simultaneous administration can be achieved viaadministration of single dosage form/fonnulation (e.g., intravenousadministration of an intravenous formulation that contains bothpharmacologically active agents). One of ordinary skill in the art candetermine through routing testing whether two given pharmacologicalcomponents are compatible together and in a given formulation.

The presently described method can be used to treat a patient sufferingfrom any condition that can be remedied or prevented by this method.Exemplary conditions are cancers, such as, fibrosarcoma, myxosarcoma,liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell cancer, basal cellcancer, adenocarcinoma, sweat gland cancer, sebaceous gland cancer,papillary cancer, papillary adenocarcinomas, cystadenocarcinoma,medullary cancer, bronchogenic cancer, renal cell cancer, hepatoma, bileduct cancer, choriocarcinoma, seminoma, embryonal cancer, Wilms' tumor,cervical cancer, testicular cancer, lung cancer, small cell lung cancer,bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma,retinoblastoma and leukemias.

All articles, books, patents, patent publications and other publicationsreferenced herein are incorporated by reference in their entireties. Inthe event: of an inconsistency between the teachings of thisspecification and the art incorporated by reference, the meaning of theteachings and definitions in this specification shall prevail(particularly with respect to terms used in the claims appended herein).For example, where the present application and a publicationincorporated by reference defines the same term differently, thedefinition of the term shall be preserved within the teachings of thedocument from which the definition is located.

EXPERIMENTAL

It is to be understood that while the invention has been described inconjunction with certain preferred and specific embodiments, theforegoing description as well as the examples that follow are intendedto illustrate and not limit the scope of the invention. Other aspects,advantages and modifications within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.

Anti-CTLA-4 antibody referenced in Examples 2-7 corresponds toanti-mouse CLTA-4 antibody purified from UC10-4F10-11 hybridoma cellline (ATCC). The cells were maintained in PFHM-II medium (Invitrogen)between the density of 1×10⁵ and 1×10⁶ cells/mL. Prior to purification,the medium was centrifuged to remove the cells. The pH of the medium wasadjusted to 8 with NaOH, and its conductivity was lowered to 7 mS/cm bydiluting with water. The pH/conductivity adjusted medium was loaded ontoa Q-FF column, and the anti-mouse CLTA-4 antibody was eluted using aNaCl gradient.

The affinity of anti-CTLA-4 to its antigen was determined by SurfacePlasmon Resonance (Biacore) and determined to be 20 pM.

Anti-PD-1 was purchased from BioXcell. It was obtained as a solutionwith the following characteristics: concentration: 4.76 mg/mL;endotoxin: <0.63 EU/mg; formulation: PBS pH 7; purity: >95%; isotype:rat IgG2a; and extinction coeff: 1.33. The affinity of anti-PD-1 to itsantigen was measured by Surface Plasmon Resonance (Biacore) and found tobe 1 nM. The purchased antibody was checked for purity by SDS-PAGE andSEC-HPLC and determined to be sufficiently pure for efficacy studieswithout further workup.

EXAMPLE 1 PEGvlation of rIL-2 with mPEG2-C2-fmoc-20K-NHS

PEGylation of rIL-2 with mPEG2-C2-fmoc-20K-NHS was previously reportedin Example 2 of WO 2012/065085. There, the synthesis was reported toresult in a mixture of 4-mers, 3-mers, 2-mers and 1-mers. Furtheranalysis of the reaction, however, revealed higher degrees of attachment(e.g., 5-mers, 6-mers and 7-mers) were also produced.

The present synthesis represents a scaled up approach for PEGylatingIL-2 with mPEG2-C2-fmoc-20K-NHS.

Purified rIL-2 (106.4 mL) at 1.44mg/ml was charged into a first vesselfollowed by the addition of 53.6 mL of formulation buffer (10 mM sodiumacetate, pH 4.5, 5% trehalose). The pH was measured at 4.62 thetemperature was measured at 21.2° C. The PEG reagent,C2-PEG2-FMOC-NHS-20K (available as described in WO 2006/138572) (13.1g), was charged into a second vessel followed by the addition of 73.3 mLof 2 mM HCl. The resulting solution was swirled by hand for 25 minutes.Sodium borate (0.5 M, pH 9.8) was added to the first vessel to raise thepH to about 9.1 and then the second vessel containing the PEG reagentwas added to the first vessel over one to two minutes. A rinse step wasthen performed by charging 8.1 mL of 2 mM HCl into the second vessel andadded to the first vessel. In the conjugation reaction, the final rIL-2concentration was 0.6 mg/rnL, the sodium borate concentration was 120mM, the pH was 9.1 +/−0.2, the temperature was 20-22° C., and the molarratio of PEG reagent to rIL-2, after adjustment for activity of thereagent (substitution level) is 35:1. The conjugation reaction wasallowed to proceed for thirty minutes and then was stopped with anacidification reaction using 75 mL of 2N acetic acid (where pH drops to4.01). The product of the reaction was diluted with water and thediluted PEGylated rIL-2 solution was filtered using a 0.2 micron filterand the filtered product is placed in sterile containers.

Thereafter, the diluted PEGylated rIL-2 solution was purified by loadingthe solution onto a chromatography column packed with SP sepharose FFresin (GE Healthcare). Following a washing step, the PEGylated rIL-2 areeluted using a sodium chloride gradient. Fractions containing 1-mers,2-mers or 3-mers are eliminated while fractions containing 4-mers,5-mers, 6-mers, 7-mers and any higher degrees of PEGylation are pooled,thereby resulting in a composition having primarily 4-mers, 5-mers and6-mers (wherein 8-mers and higher degrees of PEGylation were found to beremoved diming a washing step associated with chromatography). Thiscomposition is the one used in connection with Examples 2-6 andreferenced therein as “RSLAIL-2.”

Yields of 4-mers, 5-mers and 6-mers were found to be increased (with aconcomitant decrease in 1-mers, 2-mers and 3-mers) using the approachdescribed in this example.

EXAMPLE 2 Evaluating the Efficacy of RSLAIL-2 in Combination withAnti-CTLA-4 Antibody on the CT26 Tumor Model in Female BALB/c Mice

The objective of this study was to evaluate the antitumor activity ofRSLAIL-2 in combination with Anti-CTLA-4 antibody in the CT26 murinecolon carcinoma tumor model in female BALB/c mice.

There were 5 groups with 12 animals in each group. Included were avehicle control group treated on days 0, 4, 9 and 14, two single agentgroups (Anti-CTLA-4 antibody treated on days 0, 4, 9, 14 and 18 orRSLAIL-2 on day 0 and 9) and two combined immunotherapy groups(Anti-CTLA-4 antibody plus RSLAIL-2) where Anti-CTLA-4 antibody (givenon days 0, 4, 9, 14 and 18) treatment for one group was initiated at thesame time as RSLAIL-2 (given on day 0, 4 and 9) and for the other, fourdays prior to RSLAIL-2 treatment initiation (given on days 4, 13 and22). Treatment initiation for the study was performed 7 days afterinoculation of CT26 cells at 2×10⁶ cells/site at 0.1 mL injectionvolume. The tumor cells were injected subcutaneously in the abdominalarea. The animals were distributed accordingly based on therandomization generated by the StudyLoge software. The mean tumorvolumes on treatment day (Day 0) ranged from 111±9 mm³ to 115±10 mm³(Mean±SEM).

Tumor volumes (in mm³) and body weights (in grams) were monitored 2 to 3times a week and are presented in FIGS. 1 and 2, respectively, withdetails on the treatment schedule. The corresponding relative tumorvalues, standardized values calculated against individual tumor volumesat the start of the study and presented as percent growth are summarizedin FIG. 1 (Day 0 to Day 30). Tumor free animals were monitored for tumorregrowth for 106 days andi)ody weights were taken from treatmentinitiation.

Comparison of tumor volumes between vehicle control animals and treatedanimals on day 11 (last day the vehicle control animals were present) byOne-way ANOVA with Tukey's post-test (GraphPad Prism version 6.03 forWindows, GraphPad Software, San Diego Calif.) showed all the treatmentgroups were significantly different from the vehicle control group.

Mean tumor growth inhibition (%TGI) was assessed on Day 11 (last daycontrol animals were present) by using the following formula:

% TGI=(1−(Relative Tumor Volume (%)^(Treatment Group)÷Relative TumorVolume (%)^(Control Group))×100

There was a 53% mean inhibition for Anti-CTLA-4 antibody (Gr. 2) treatedtumors and 58% for the RSLAIL-2 group (Gr. 3). Combined immune therapyco-administration (RSLAIL-2 and Anti-CTLA-4 antibody initiated at thesame time on Day 0) yielded 74% inhibition. Combined immune therapy withAnti-CTLA-4 antibody initiated on Day 0 and RSLAIL-2 treatment initiatedon Day 4 (Gr. 5) yielded the greatest inhibition among the treatments at88%.

One of the twelve (1/12) animals in group 4 were tumor free by day 14. Atotal of 4 animals were tumor free by day 28. In group 5, two (2/12)animals were tumor free by day 14 and a total of 8 animals were tumorfree by day 25. See Table 1. All these animals from group 4 and 5remained tumor free until study termination. (106 days from treatmentinitiation).

TABLE 1 Tumor Volumes (Mean ± SEM in mm³) Tumor Mean Volume on TumorTreatment Tumor Growth Initiation Volume on Inhibition Mean (Day 0) Day11 (%) Tumor Tumor Free (MEAN ± (MEAN ± (Endpoint: Delay Animals onTreatment Dose N SEM) SEM) 18 Days) (Days) Termination 1. VehicleControl NA 12 111 ± 9  1499 ± 196 NA 1¹ of 12 (Day 18) 2. Anti CTLA-4100 ug 12 115 ± 10  732 ± 165 53.3 1¹ of 12 Antibody (Day 18) 3.RSLAIL-2 0.8 mg/kg 12 111 ± 11  600 ± 143 58.4 1¹ of 12 (Day 18) 4.RSLAIL-2 (Day 0) + 100 ug 12 113 ± 12  414 ± 115 74.2 4 of 12ANTI-CTLA-4 0.8 mg/kg (Day 106) ANTIBODY (Day 0) 5. RSLAIL-2 (Day 4) +0.8 mg/kg 12 111 ± 9  170 ± 27 88.1 8 of 12 ANTI-CTLA-4 100 ug (Day 106)ANTIBODY (Day 0) ¹The group (including this animal) was collectivelyremoved from the study on Day 18

Mean body weights ranged from 17.4±0.3 g to 18.2±0.3 g (Mean±SEM) ontreatment day. No significant mean body weight loss below baselineduring the, treatment phase was observed from any of the treatmentgroups (FIG. 3).

EXAMPLE 3 Evaluating the Efficacy of RSLAIL-2 in Combination withAnti-CTIA-4 Antibody on the EMT6 Tumor Model in Female BALB/c Mice

The objective of this study was to evaluate the antitumor activity ofRSLAIL-2 in combination with Anti-CTLA-4 antibody on the EMT6 murinemammary carcinoma tumor model in female BALB/c mice.

There were 5 groups with 12 animals each in each group. Included were avehicle control group treated on days 0, 4, 9 and 14, two single agentgroups (Anti-CTLA-4 antibody treated on days 0, 4, 9, 14 and 18 orRSLAIL-2 on day 0 and 9) and two combined immunotherapy groups(Anti-CTLA-4 antibody plus RSLAIL-2) where Anti-CTLA-4 antibody (givenon days 0, 4, 9, 14 and 18) treatment for one group was initiated at thesame time as RSLAIL-2 (given on day 0, 4 and 9) and for the other, fourdays prior to RSLAIL-2 treatment initiation (given on days 4, 13 and22). Treatment initiation for the study was performed 7 days afterinoculation of EMT6 cells at 2×10⁶ cells/site at 0.1 mL injectionvolume. The tumor cells were injected subcutaneously in the abdominalarea. The animals were distributed accordingly based on therandomization generated by the StudyLog® software. The mean tumorvolumes on treatment day (Day 0) ranged from 144 8 mm³ to 147±10 mm³(Mean±SEM).

Tumor volumes (in mm³) and body weights (in grams) were monitored 2 to 3times a week and tumor volume data are presented in FIG. 4 (Day 0 to Day30) with details on the treatment schedule. Tumor-free animals weremonitored for tumor regrowth for 106 days, from treatment initiationwhich is presented in FIG. 5.

Comparison of tumor volumes between vehicle control animals and treatedanimals on day 18 (last day the vehicle control animals were present) byOne-way ANOVA with Tukey's post-test (GraphPad Prism version 6.03 forWindows, GraphPad Software, San Diego Calif.) showed a significantdifference between the vehicle controls (group 1) versus group 2(Anti-CTLA-4 antibody treatment) and group 5 (Anti-CTLA-4 antibodystarted on Day 0 +RSLAIL-2 started on Day 4). No statistical differencewas noted between group 1 (vehicle controls) versus group 3 (RSLAIL-2alone) and group 4 (RSLAIL-2+Anti-CTLA-4 antibody both treatmentsinitiated on day 0).

Mean tumor growth inhibition (% TGI) was assessed on Day 18 (last daycontrol animals were present) by using the following formula:

% TGI=(1−(Relative Tumor Volume (%)^(Treatment Group)÷Relative TumorVolume (%)^(Control Group))×100

There was a 55% mean inhibition for Anti-CTLA-4 antibody (Gr. 2) treatedtumors and 22% for the RSLAIL-2 group (Gr. 3). Combined immune therapyco-administration (RSLAIL-2 and Anti-CTLA-4 antibody initiated at thesame time on Day 0) yielded 27% inhibition. Combined immune therapy withAnti-CTLA-4 antibody initiated on Day 0 and RSLAIL-2 treatment initiatedon Day 4 (Gr. 5) yielded the greatest inhibition among the treatments at92%. The tumor on one animal in the vehicle control group was observedto have completely autoregressed by day 11. In spite of this, mean tumorvolume was 1789 mm³±196 (MEAN±SE, N=12) by Day 18 when the group wascollectively removed from the study. One of the twelve animals in group4 was also tumor free by day 18. Mean tumor volume for the rest of thegroup was 1361±214 mm³ when the whole group was removed from the study(on Day 18).

Five of the twelve (5/12) animals in group 5 were tumor free by day 14.A total of 10 animals were tumor free by day 18. All 10 animals remainedtumor free until termination of the study (106 days from treatmentinitiation). See Table 2.

TABLE 2 Tumor Volumes (Mean ± SE in mm³) Tumor Mean Volume on TumorTreatment Tumor Growth Initiation Volume on Inhibition (Day 0) Day 18(%) Tumor Free (MEAN ± (MEAN ± (Endpoint: Animals on Treatment Dose NSEM) SEM) 18 Days) Termination  6. Vehicle Control NA 12 147 ± 10 1789 ±196 NA 1¹ of 12 (Day 18)  7. Anti CTLA-4 100 ug 12 145 ± 9   824 ± 18555 None  8. RSLAIL-2 0.8 mg/kg 12 144 ± 11 1405 ± 13  22 None  9.RSLAIL-2 (Day 0) + 100 ug 12 145 ± 10 1361 ± 214 27 1¹ of 12 ANTI-CTLA-40.8 mg/kg (Day 18) ANTIBODY (Day 0) 10. RSLAIL-2 (Day 4) + 0.8 mg/kg 12144 ± 8  155 ± 83 92 10 of 12 ANTI-CTLA-4 100 ug (Day 107) ANTIBODY (Day0) ¹The group (including this animal) was collectively removed from thestudy on Day 18

Mean body weights ranged from 17.8±0.3 g to 18.4±0.4 g (Mean±SEM) ontreatment day (FIG. 6). No significant body weight loss was observedfrom any of the treatment groups.

EXAMPLE 4 Evaluating the Efficacy of RSLAIL-2 in Combination withAnti-CTLA-4 Antibody Compared to that of Proleukin with Anti-CTLA-4Antibody Treatment on the EMT6 Tumor Model in Female BALB/c Mice

The objective of this study is to evaluate the antitumor activity ofRSLAIL-2 in combination with Anti-CTLA-4 antibody and compare it to thatof Proleukin on the EMT6 murine mammary carcinoma tumor model in femaleBALB/c mice.

There were 7 groups with 10 animals each. Included were an AntibodyControl (Gr. 1) given on days 0, 4, 8 and 13) and three single agentgroups. These were Anti-CTLA-4 antibody (Gr. 2) given on days 0, 4, 8,13 and 18. Proleukin (Gr. 3) given from day 0 to 4 then 7 to 11 and aRSLAIL-2 (Gr. 4) given on day 0 and 9. Also included were three combinedimmune therapy groups. A combined immunotherapy group using Anti-CTLA-4antibody with Proleukin (Gr. 5) and 2 combination treatment group usingAnti-CTLA-4 antibody with RSLAIL-2 (Gr. 6 and 7) on different treatmentschedules. Anti-CTLA-4 antibody regimen was initiated on day 4 and givenagain on days 9 and 13 while RSLAIL-2 was initiated on day 0 and givenagain on day 9 for group 6. Anti-CTLA-4 antibody therapy for group 7 wasinitiated on day 0 and given again on days 4, 8 and 13 while RSLAIL-2was initiated on day 4 and given again on days 13 and 22. Treatmentinitiation (Day0) was designated as 7 days after inoculation of EMT6cells at 2×10⁶ cells/site at 0.1 mL injection volume on the mice. Thetumor cells were injected subcutaneous in the abdominal area. Theanimals were distributed accordingly on Day 0 based on the randomizationgenerated by the StudyLog® software. The mean tumor volumes on treatmentday ranged from 159±7 mm³ to 170±8 mm³ (Mean±SE).

Tumor volumes (in mm³) and body weights (in grams) were monitored 2-3times a week and are presented in the FIGS. 7 and 8, respectively (Day 0to Day 28). Tumor Free animals were monitored regrowth and healthconditions for 99 days (from treatment initiation).

Comparison of tumor volumes between control animals and treated animalson day 18 (last day the vehicle control animals were present) by One-wayANOVA with Tukey's post-test (GraphPad Prism version 6.03 for Windows,GraphPad Software, San Diego Calif.) showed that only group 5(Anti-CTLA-4 antibody +Proleukin) and group 7 (Anti-CTLA-4 antibody+RSLAIL-2) were significantly different from those of controls. However,although significantly different from controls, mean tumor volume of thetwo treatment groups were not significantly different from one another.

Mean tumor growth inhibition (% TGI) was assessed on Day 18 (last daycontrol animals were present) by using the following formula:

% TGI=(1−(Relative Turrior Volume (%)^(Treatment Group)÷Relative TumorVolume (%)^(Control Group))×100

There was a 27% mean inhibition for Anti-CTLA-4 antibody treatment alone(Gr. 2). Inhibition of tumor growth was 24% by Proleukin (Gr. 3) and 5%by RSLAIL-2 (Gr. 4), combined immune therapy with Anti-CTLA-4 antibodyand Proleukin (GR 5) yielded 26% inhibition, RSLAIL-2 with Anti-CTLA-4antibody treatment (Gr. 6) gave a 2% inhibition while group 7(Anti-CTLA-4 antibody with RSLAIL-2) yielded a 94% inhibition.

Several animals were observed to be tumor free by day 18. There werethree (3/10) from group 5, one (1/10) from group 6 and five (5/10) ingroup 7. By study termination (Day 99), the total number of tumor freeanimals were from groups 5, 6 and 7 were 4/10, 1/10 and 7/10respectively. See Table 3.

TABLE 3 Tumor Volumes (Mean ± SE in mm³) Tumor Volume Tumor Mean Tumoron Treatment Volume on Growth Initiation Day 18 Inhibition (%) TumorFree Treatment (Day 0) (MEAN ± (Endpoint: 18 Animals on Group Dose N(MEAN ± SEM) SEM) Days) Termination 1. Antibody 100 ug 10 166 ± 6 1803 ±174 NA NA Control (Day 0, 4, 8, 13) 2. Anti CTLA-4 100 ug 10 166 ± 71294 ± 122 27 None antibody (Day 0, 4, 8, 13) 3. Proleukin 0.5 10 165 ±7 1335 ± 236 24 None (Day 0-4, 7-11) mg/kg 4. RSLAIL-2 0.8 10 167 ± 81040 ± 78  5 None mg/kg 5. Anti-CTLA-4 0.8 10 159 ± 7 155 ± 83 76 4 of10 antibody (Day 0) + mg/kg (Day 99) Proleukin 0.5 (Day 4) mg/kg 6.RSLAIL-2 (Day 0.8 10 165 ± 7 1701 ± 229 2 1 of 10 0) + mg/kg (Day 99)Anti-CTLA-4 100 ug antibody (Day 4) 7. Anti-CTLA-4 100 ug 10 170 ± 8 108± 56 94 7 of 10 antibody (Day 0.8 (Day 99) 0) + mg/kg RSLAIL-2 (Day 4)

EXAMPLE 5 Efficacy Study Testing RSLAIL-2 in Combination withAnti-CTLA-4 Antibody on the EMT6 Tumor Model in Female BALB/c Mice (Day0, 3 and 11 Sample Collection for Flow Cytometry Analysis)

Prior combination RSLAIL-2 and Anti-CTLA-4 antibody efficacy studiessuggest synergy in the EMT6 mouse mammary model. The objective of thisstudy was to evaluate/assess the antitumor activity of RSLAIL-2 incombination with Anti-CTLA-4 antibody on the EMT6 murine mammarycarcinoma tumor model in female BALB/c mice.

In addition, in order to identify immune population responsible forcombinatorial efficacy, tissue (tumor and spleen) was collected andprocessed at 0, 3 and 11 days after treatment initiation. Identificationand quantification of immune cells that infiltrated the tissues wasevaluated by flow cytometry analysis. The results were compared betweentreatment groups.

In-vivo phase

There were 6 treatment groups in this study with 3-10 animals in eachgroup. Included was an Antibody control, IgG2a (Group 1), given at 100ug/mouse on day 0, 4, and 8; an Anti-CTLA-4 antibody treatment (Group2), given at 100 ug/mouse on day 4 and 8; Proleukin treatment (Group 3),given at 0.5 mg/kg on days 4 to day 8; and RSLAIL-2 treatment (Group 5),given a single administration at 0.8 mg/kg on day 4. Also included werethree groups to be treated with a combination of Anti-CTLA-4 antibody(100 ug/mouse given on day 0, 4, and 8) and Proleukin (0.5 mg/kg givenon day 4 to day 8), Group 4 or RSLAIL-2 (0.8 mg/kg given on day 4),Group 6.

Treatment was initiated 7 days after the animals were inoculated with2×10⁶ cells/site of EMT6 murine mammary carcinoma cells (0.1 mL)subcutaneously, in the abdominal region. The mice were distributed intotreatment groups according to tumor volumes.

The animals were sacrificed and tissue (tumor and spleen) were collectedat Day 0 (Naïve Controls), Day 3 (Group 1 and 6), group 6 day 3 animalshad been dosed with only Anti-CTLA-4 antibody at this time, addition ofRSLAIL-2 begin at day 4, samples were also taken at Day 11 (all groups)after treatment initiation. Three animals were selected for eachcollection day. The animals selected had intact tumors (non-necrotic)and volumes that best approximated the group mean tumor volume for thatday.

Mean tumor volume was 164±10 mm³ for naïve group on treatment day 0. Onday 3, mean tumor volumes were 282±7 mm³ and 333±26 mm³ (Mean±SEM),respectively for Group 1 and Group 6. On day 11, mean tumor volumes were843±138 mm³ for Group 1, 1059±135 mm³ for Group 2, 814±70 mm³ for Group3, 832±262 mm³ for Group 4, 620±103 mm³ for Group 5, and 255±11 mm³ forGroup 6 (Mean±SEM). See FIG. 9.

Animals were weighed at least once a week. The mean body weights were18.9±0.3, 18.6±0.3, 19.2±0.2, 18.7±0.2 17.8±0.3, 18.9±0.3 grams forGroups 1, 2, 3, 4, 5, and 6, respectively, on treatment day (Day 0). OnDay 3, the mean body weights for Group 1 and 6 were 19.1±0.7 and 19±0.3grams respectively. Mean body weights on Day 11 were 19.8±0.2, 19.4±0.2,19.9±0.2, 20±0.5, 19.6±0.4 and 19.5±0.4 grams (Mean±SEM) for groups 1,2, 3, 4, 5, and 6 respectively. See FIG. 10.

Ex-Vivo Phase

Collected tissue samples were manually minced using a scalpel followedby a 13 minute enzymatic digestion incubated at 37° C. The components ofthe digestion buffer were of 2.5 mg/ml Collagenase Type II (GIBCO BRL),2.5 mg/ml Collagenase Type IV (GIBCO BRL), and 0.5 mg/ml DNase(Sigma-Aldrich) in PBS/BSA. After incubation, the digest was quenched byadding Waymouth's MB (GIBCO BRL) containing 10% FBS (heat inactivated;GIBCO BRL) and filtered through a 70-uM nylon filter (Falcon) thatyielded a single cell suspension. The cells were washed in HBSS andcentrifuged, then resuspended in fresh HBSS. An aliquot was taken fromeach sample for counting then stained with eFlour-450 viability dye. Thesamples were then plated in a 96-deep well plate, stained and collectedfor flow cytometry analysis.

The tumor and spleen cell samples were first treated with a fixableviability indicator and then stained for viable immune cells, CD3, CD4,CD8, CD25, CD44, CD122, Foxp3 (internal stain), DX5 and NKp46 surfaceantigens.

The total amount of live immune cells were counted for each of thesamples (3 per treatment group) and used for gating/collecting totalevents for CD4+, TREG+ cell, CD8+, Memory effector CD8+ cells, total NKcells, mature NK cells (which were normalized to 1 cubic millimeter oftumor tissue) as well as the total cell counts for the spleen for eachof the treatment groups. The primary counts were derived from thesummarized raw data of the flow cytometer reading and analyzed usingFlowJo.

Flow Cytometry

Three naïve animals were sacrificed on day 0. Three animals weresacrificed from Group 1 (having received isotype control) and from Group6 (having received Anti-CTLA-4 antibody treatments only) on day 3, andthree animals from all Groups 1-6 were sacrificed on day 11 after theinitiation of treatment.

The graphical representation of the data can be found in FIG. 11 andFIG. 12 for Day 11 in tumor and spleen, respectively.

There is no difference in CD4+ or regulatory T cell population (from Day3-Day 11) when treated with Anti-CTLA-4 antibody alone. Pre-treatmentwith Anti-CTLA-4 antibody followed by Proleukin or RSLAIL-2 treatmentincreases CD4+population; however, there is no difference in regulatoryT cell population (Anti-CTLA-4 antibody vs.

Anti-CTLA-4 antibody+ RSLAIL-2 or Proleukin). There is no difference inCD4+ cells in RSLAIL-2+Anti-CTLA-4 antibody vs. RSLAIL-2 alone; however,RSLAIL-2+Anti-CTLA-4 antibody reduces regulatory T cell population whencompared to RSLAIL-2. There is a large population of CD4+ cells in theRSLAIL-2+Anti-CTLA-4 antibody group that are not accounted for; theseresults suggest that the majority of these cells are not Tregs.

Pre-treatment with Anti-CTLA-4 antibody followed by RSLAIL-2 treatmentsignificantly increases CD8+ population, P=0.0078, and memory effectorCD8 cells, P=0.0058. (Two tailed t test Anti-CTLA-4 antibody+RSLAIL-2vs. Anti-CTLA-4). In addition, RSLAIL-2+Anti-CTLA-4 antibodysignificantly increase CD8+ and memory effector CD8 cells when comparedto RSLAIL-2 alone.

Anti-CTLA-4 antibody alone had no trending effect on total NK cellpopulation; however, mature NK cells were significantly decreased(P=0.0278 from day 3 to day 11). Pretreatment with Anti-CTLA-4 antibodyfollowed by RSLAIL-2 treatment increases total NK population and matureNK cell population. No difference in mature NK cells inRSLAIL-2+Anti-CTLA-4 antibody group when compared to RSLAIL-2 group.

EXAMPLE 6 Efficacy of Combined Immune Therapy with RSLAIL-2 andAnti-PD-1 Antibody on CT26 Murine Colon Carcinoma Tumor Growths inFemale BALB/c Mice

The objective of this study was to evaluate the antitumor activity ofcombined immune therapy with RSLAIL-2 and Anti-PD-1 antibody on the CT26murine colon carcinoma tumor model in female BALB/c mice.

There were 4 groups with 10 animals each. Included were a vehicleControl group (Gr. 1), an Anti-PD-1 antibody treatment group, anRSLAIL-2 treatment group (Gr. 3) and a. combination treatment grouptreated first with Anti-PD-1 antibody (200 ug) on days and RSLAIL-2 (0.8mg/kg) 4 days after. CT26 cells at 2×10⁶ cells/site in 0.1 mL injectionvolume were implanted on the mice subcutaneously in the abdominal area.Treatment was initiated seven days after tumor cell inoculation (Day 0).The animals were distributed accordingly on Day 0 based on therandomization generated by the StudyLoge software. The mean tumorvolumes on treatment day (Day 0) ranged from 123±5 mm³ to 127±6 mm³(Mean±SE).

Tumor volumes (in mm³) and body weights (in grams) were monitored 2 to 3times a week and are presented in the FIGS. 13 and 14, respectively. Thestudy was monitored for 16 days.

Comparison of tumor volumes between control animals and treated animalson day 12 (last day the vehicle control animals were present) by One-wayANOVA with Tukey's post-test (GraphPad Prism version 6.03 for Windows,GraphPad Software, San Diego Calif.) showed that tumor volumes for allthe treatment groups were significantly different from those ofuntreated controls.

Percent Tumor Growth Inhibition (% TGI) was calculated using thefollowing formula:

% TGI=(1−(Relative Tumor Volume (%)^(Treatment Group)+Relative TumorVolume (%)^(Control Group))×100

There was a 55% and a 58% mean tumor growth inhibition observed withAnti-PD-1 antibody (Gr. 2) and RSLAIL-2 (Gr. 3) respectively. Tumorgrowth inhibition for the group given both treatments in combination(Gr. 4) was observed at 83%. Five (5/10) animals in group 4 at studytermination had tumors with volumes less than their initial volumes onDay 0.

Mean Tumor Volume Quadrupling Time (TVQT), time in days it takes tumorsto grow to 4 times their initial volume was interpolated usingnon-linear second degree polynomial analysis (GraphPad Prism version6.03 for Windows, GraphPad Software, San Diego Calif.) to assess TumorGrowth Delay (TGD). Mean tumor volume quadrupling time for controltumors was 5.2 days, 7.6 days for Group 3 (Anti-PD-1 antibody), 8.2 daysfor Group 3 (RSLAIL-2) and 15.6 for the combined immune therapy group(Gr. 4). Mean tumor groWth delay for Group 2, Group 3 and Group 4 are2.4 days, 3.0 days and 10.4 days, respectively. See Table 4.

TABLE 4 Tumor Volumes (Mean ± SE in mm³) Tumor Volume on Mean TumorTreatment Tumor Growth Inhibition Mean Mean Initiation Volume on (%)Group Group (Day 0) Day 12 (Endpoint: 12 TVQT TGD Treatment Dose N (MEAN± SE) (MEAN ± SE) Days) (Days) (Days) 1. Antibody 200 ug 10 125 ± 5 1983± 248  NA 5.2 NA Control (Day 0, 4, 8, 13) 2. Anti PD1 200 ug 10 126 ± 5897 ± 82  55 7.6 2.4 antibody (Day 0, 4, 8, 13) 3. RSLAIL-2 0.8 10 127 ±6 833 ± 176 58 8.2 3.0 (Day 0, 9) mg/kg 4. Anti PD1 200 ug 10 123 ± 5334 ± 109 83 15.6 10.4 antibody 0.8 (Day 0, 4, 8, 13) + mg/kg RSLAIL-2(Day 0, 9)

Mean body weights ranged from 17.5±0.2 g to 18.1±0.2 g (Mean±SEM) on Day0 (FIG. 13). No significant body weight loss was observed (FIG. 14)except for one animal in Group 2 which was removed from the study on day12. Necropsy revealed a metastatic lesion in the pulmonary cavity.

Greater tumor growth inhibition (TGI) and tumor growth delay (TGD) wasobserved in the animals given a combination of Anti-PD-1 and RSLAIL-2than those treated with either single agent.

EXAMPLE 7 Re-Challenge Study Re-Challening Tumor-Free Animals with EMT6Murine Mammary Carcinoma Tumors After Effective Combined Immune Therapywith RSLAIL-2 and Anti-CTLA-4

The objective of this example was to evaluate the extent and duration ofefficacy when treating mice implanted with EMT6 mammary carcinoma tumorswith combined immune therapy using RSLAIL-2 and a commercially availablerodent Anti-CTLA-4 checkpoint blockade antibody. As previouslydemonstrated in Examples 2-6, this combination yields significant tumorfree animals and by re-challenging tumor-free animals with EMT6 tumorcells or CT26 tumor cells, the ability of this combination to elicittumor-specific responses are evaluated.

This re-challenge study was conducted in three phases.

The initial part of the study (“Phase I”) used 80 female BALB/c micebearing EMT6 (Murine Mammary Carcinoma) tumors. Ten (10/80) animals wererandomly selected and assigned to the antibody control group (100 μg onDays 0, 4, 9, 13 i.p.). The rest of the animals (70/80) were treatedwith a combination of Anti-CTLA-4 i.p. (100 μg on Days 0, 4, 9, 13 and18) and RSLAIL-2 i.v. (0.8 mg/kg on Days 4, 13 and 22).

Treatment initiation (Day 0) was designated as seven days afterinoculation of EMT6 cells at 2×10⁶ cells/site in 0.1 mL injectionvolumes, injected subcutaneously in the abdominal area. The mean tumorvolumes on treatment initiation were 206±15 and 222±8 (MEAN±SE) for thevehicle control and treatment group respectively.

Phase I was monitored until Day 48. Tumor volumes (in mm³) and bodyweights (in grams) were measured 2 to 3 times a week and presented inFIG. 15. The corresponding relative tumor volumes, standardized valuescalculated against individual tumor volumes at the start of the study(Day 0) and presented as percent growth and the relative changes in bodyweight are summarized in FIG. 16.

The results from Phase I of the study showed a Tumor Growth Inhibition(TGI) of 73% (P<0.0001), calculated using mean relative tumor volumesbetween control animals (698%) and treated animals (189%) on day 14(last day all the control animals were present) and then compared usingunpaired t-test with Welches correction factor (GraphPad Prism version6.03 for Windows, GraphPad Software, San Diego Calif.).

The Mean Tumor Growth Delay (TGD) of the animals (N=39) in Phase I thatdid not exhibit complete response to the combined immune therapy was12.7 days (P<0.0001). Tumor Volume Quadrupling Time (TVQT) was computedby using interpolated individual tumor time to 400% growth with secondorder polynomial non-linear regression analysis (GraphPad Prism).Tumor-free animals (complete responders) were not included in theassessment for TGD.

In Phase I, treated animals were observed with rough coats (slight)around 5 days after the first and second RSLAIL-2 administration. Roughcoats were not observed after the third administration. No significantbody weight losses were observed (FIG. 16).

A summary of the results from Phase I of the study are provided in Table5.

TABLE 5 Phase I Summary of Results (Mean ± SE) Tumor Mean Mean VolumeRelative Tumor Tumor on Tumor Growth Tumor Free Treatment VolumeInhibition Growth Animals Initiation on Day 14 (%) TVQT Delay by DayTreatment Dose N (mm³) (%) On Day 14 (Days) (Days) 48 Antibody ControlNA 10 206 ± 15 698 ± 60 NA 9.7 NA NA (Day 0, 4, 8, 13) RSLAIL-2 0.8mg/kg 70 222 ± 8 189 ± 25 73 ¹22.4 ¹12.7 31 (Day 4, 13, 22) + 100 ug (P< 0.0001) (P < 0.0001) (44%) Anti CTLA-4 (Day 0, 4, 9, 13, 18) ¹Onlyinterpolated for animals with partial response, animals with completeresponse (i.e. tumor free) were not included ²Mean tumor growthinhibition (% TGI) was assessed on Day 18 (last day control animals werepresent) by using the formula: % TGI = (1 − (Relative Tumor Volume(%)^(Treatment Group) ÷ Relative Tumor Volume (%)^(Control Group)) × 100

The next part of the study, (“Phase II”) was initiated on Day 49. Atotal of 40 animals (10 age-appropriate naïve animals and 30 animalsthat completely responded to the RSLAIL-2+Anti CTLA-4 combinedimmune-therapy) were used for 3 groups and distributed as described inTable 6.

TABLE 6 Phase II Study Design ¹Group N Age Appropriate Naïve AnimalsChallenged with EMT6 Cells 10 EMT6 Tumor-Free Animals Re-challenged withCT26 10 EMT6 Tumor-Free Animals Re-challenged with EMT6 20 ¹Tumor FreeAnimals refer to EMT6 tumor bearing animals from Phase I that completelyresponded to the RSLAIL-2 + Anti CTLA-4 therapy

On Day 49, the animals were implanted with either EMT6 or CT26 tumorcells (2×10⁶ in 0.1 mL media), injected subcutaneously in the abdominalregion.

The results from Phase II of the study showed tumor uptake and growth onthe animals were evident five days after inoculation (Day54). All theanimals in Group 1 (Age Appropriate Naive) and Group 2 (EMT6 tumor-freeanimals re-challenged with CT26) were bearing tumors with mean volumesof 167±22 and 177±11 respectively (MEAN±SE). Only 85% tumor uptake ratewas observed in Group 3 (EMT6 Tumor Free Animals Re-challenged withEMT6) five days after inoculation. Tumor volumes were observed to berelatively smaller than those on in the other groups. Mean tumor volumewas 62±8 mm³. Fourteen of the twenty (70%) animals in this group weretumor free (completely rejected EMT6 tumor implants) 17 clays afterre-challenge and remained tumor free until the end of Phase II (Day109).

Animals in Group 2, EMT6 tumor free animals after combinedimmune-therapy in Phase I and re-challenged with CT26 tumors, had a meantumor volume of 1257±201 (Mean±SE) 14 days after re-challenge and a meantime to grow to 1000 mm³ volume of 13.6±0.8. Tumor growth did not appearto be affected by Phase I therapy which implies tumor-type specificityof the immune response. See the summary of the results from Phase II ofthe study provided in Table 7.

One animal from Group 1 was found expired on day 14. Necropsy revealedwhat appeared to be metastatic lesions in the lungs (not verified byhistology) and bloody fluid in the chest cavity. Other than this, noother clinical observations or significant body weight loss was noted.See FIG. 17.

Mean tumor volumes (FIG. 18) between the Age Appropriate Naïve animals(1541±144 mm³) and the EMT6 tumor free animals re-challenged with EMT6(192 ±107) were compared 21 days after implantation to assess TGI(87.5%).

TVQT could not be assessed due to a lack of baseline tumor volumestarting point. In its place, time for tumor growth to 1000 mm3 volumefor each of the tumor-bearing animals were interpolated (using secondorder polynomial non-linear regression analysis of GraphPad Prism) toaid in assessing and approximate tumor growth delay. Time (in days)taken for growth to 1000 mm³ tumor volume were 16.9±1 and 13.6±0.8(Mean+SE) days for Groups 1 and 2, respectively. Only the animals (6/20)that did not completely reject EMT6 re-challenge in Group 3 wereassessed for time to 1000 mm³ growth, which was 30.3±5.5. Mean tumorgrowth to 1000 mm³ delay for these animals was calculated to be 13.4days.

TABLE 7 Phase II Summary of Results (Mean ± SE) Mean Tumor Mean Tumor-Mean Volume 21 Tumor Free Tumor Time taken Tumor days after GrowthAnimals Volume to grow to Growth Delay tumor Inhibition by on Day 1000mm³ (Days to 1000 implantation (%) Day Treatment N 5 (Days) mm³) (mm³)On Day 21 109 Age Appropriate 10 167 ± 22 16.9 ± 1   NA 1541 ± 144  NANA Naïve Animals Challenged with EMT6 EMT6 Tumor- 10 177 ± 11 13.6 ± 0.8NA NA NA NA Free Animals Re-challenged with CT26 EMT6 Tumor- 20 62 ± 830.3 ± 5.5 13.4 192 ± 107 87.5 14 Free Animals (6/20animals) (6/20Animals) Re-challenged with EMT6

The final part of the study (Phase III) was designated over Days 109 to168. Animals (N=14) that completely rejected the EMT6 re-challenge fromPhase II were again inoculated/re-challenged (on Day 109) with 2×10⁶EMT6 tumor cells (group 2) and monitored for 59 days. Age appropriateanimals (N=5) were also inoculated for controls (group 1) at the sametime.

From the final part of the study, it was observed that tumor uptake andgrowth were apparent three days after inoculation with mean tumorvolumes of 101±14 and 69±6 for Groups 1 and 2 respectively. Animalsgiven the second re-challenge were completely tumor free (completerejection) 21 days after tumor cell implantation while all 5 of theirage appropriate counterparts had a mean tumor volume of 1550±401 mm³.Animals in Group 2 remained tumor free until the end of the study (Day168) and no significant body weight loss was observed.

This study demonstrates the efficacy of combining immune therapy usingRSLAIL-2 and Anti-CTLA 4 in EMT6 murine mammary carcinoma tumors (PhaseI) as well as durability and specificity of response, and the long termeffect of the therapy in animals that respond well to treatment (PhaseII and III). See FIG. 21 for a summary of the study from beginning toend and individual tumor growths for each phase.

EXAMPLE 8 In vivo Depletion Study Assessing The Contribution ofCytotoxic Immune Cell Populations to Anti-Tumor

Immunity Against EMT6 Murine Mammary Carcinoma Tumors After EffectiveCombined Immune Therapy with RSLAIL-2 and Anti-CTLA-4 or Anti-PD-1

The objective of this example was to assess the relative contribution ofNatural Killer (NK) cells and CD8⁺ Cytotoxic T lymphocytes to antitumorefficacy when RSLAIL-2 is combined with immune checkpoint inhibitionthrough antibody blockade of CTLA-4 or PD-1.

This study utilized 58 female BALB/c mice bearing EMT6 (Murine MammaryCarcinoma) tumors. Ten (10/58) animals were randomly selected andassigned to the vehicle control group (Days 0, 4, 8, i.p.). Eight (8/58)animals were assigned to the treatment group receiving a combination ofanti-CTLA-4 i.p. (100 μg on Days 0, 4, and 8) and RSLAIL-2 i.v. (0.8mg/kg on Day 4). Eight (8/58) animals were assigned to the CD8 depletiongroup, which in addition to the treatment of RSLAIL-2 and anti-CTLA-4,CD8 T cells were depleted by serial injection of rat anti-mouse CD8a(100 μg i.p. on Days −2, 0, 7). Eight (8/58) additional animals wereassigned to the NK depletion group which in addition to the treatment ofRSLAIL-2 and anti-CTLA-4, NK cells were depleted by serial injection ofrabbit anti-bovine Asialo-GM1 (50 μl i.p. on Days −2, 0, 7).

Eight (8/58) animals were assigned to the treatment group receiving acombination of anti-PD-1 i.p. (100 μg on Days 0, 4, and 8) and RSLAIL-2i.v. (0.8 mg/kg on Day 4). Eight (8/58) animals were assigned to the CD8depletion group, which in addition to the treatment of RSLAIL-2 andanti-PD-1, CD8 T cells were depleted by serial injection of ratanti-mouse CD8a (100 μg i.p. on Days −2, 0, 7). Eight (8/58) additionalanimals were assigned to the NK depletion group which in addition to thetreatment of RSLAIL-2 and anti-PD-1, NK cells were depleted by serialinjection of rabbit anti-bovine Asialo-GM1 (50 μl i.p. on Days −2, 0,7).

Treatment initiation (Day 0) was designated as seven days afterinoculation of EMT6 cells at 2×10⁶ cells/site in 0.1 mL injectionvolumes, injected subcutaneously in the abdominal area, and the studywas 11 days in duration. Tumor volumes (in mm³) and body weights (ingrams) were measured 2 to 3 times a week. Table 8 presents the Initialand Mean Relative Tumor Volume for each group on Day 11. Forcombinations with RSLAIL-2 and

Anti-CTLA-4, tumor volumes and body weights are presented in FIG. 22,while relative tumor volume and percent body weight change from Day 0are presented in FIG. 23. For combinations with RSLAIL-2 and Anti-PD-1,tumor volumes and body weights are presented in FIG. 24, while relativetumor volume and percent body weight change from, Day 0 are presented inFIG. 25.

The results of the study showed a Tumor Growth Inhibition (TGI) of 76.9%(p<0.05) in the RSLAIL-2 and Anti-CTLA-4 treatment group, calculatedusing Mean Relative Tumor Volumes between control animals (553%) andtreated animals (128%) on Day 11 utilizing a one-way ANOVA and Tukey'smultiple comparisons post-test (GraphPad Prism version 6.03 for Windows,GraphPad Software, San Diego Calif.).

When treatment of RSLAIL-2 and Anti-CTLA-4 was combined with serialinjections of neutralizing CD8a antibodies, leading to in vivo depletionof cytotoxic CD8 T cells, the result was abrogation of treatmentefficacy with a Mean Relative Tumor Volume of 520% and Tumor GrowthInhibition of 5.98% on Day 11 which did not reach statisticalsignificance compared to the vehicle control.

When treatment of RSLAIL-2 and Anti-CTLA-4 was combined with serialinjections of neutralizing anti-Asialo-GM1 antibodies, leading to invivo depletion of NK cells, the result was abrogation of treatmentefficacy with a Mean Relative Tumor Volume of 483% and Tumor GrowthInhibition of 12.7% on Day 11 which did not reach statisticalsignificance compared to the vehicle control.

When RSLAIL-2 was combined with Anti-PD-1, the Mean Relative TumorVolume at Day 100 was 285% resulting in a Tumor Growth Inhibition (TGI)of 48.5% (p<0.05).

When treatment of RSLAIL-2 and PD-1 was combined with serial injectionsof neutralizing CD8a antibodies, the result was abrogation of treatmentefficacy with a Mean Relative Tumor Volume of 539% and Tumor GrowthInhibition of 2.53% on Day 11 which did not reach statisticalsignificance compared to the vehicle control.

When treatment of RSLAIL-2 and Anti-PD-1 was combined with serialinjections of neutralizing anti-Asialo-GM1 antibodies, the result wasabrogation of treatment efficacy with a Mean Relative Tumor Volume of364% and Tumor Growth Inhibition of 34.2% on Day 11 which did not reachstatistical significance compared to the vehicle control.

While no significant body weight loss was observed from RSLAIL-2 whencombined with Anti-CTLA-4 or Anti-PD-1, or when the treatment wascombined with the addition of CD8 or anti-Asialo-GM1 antibody ablation,combination with anti-Asialo-GM1 led to two animals found expired on Day8 of the study in the group also receiving RSLAIL-2 and Anti-CTLA-4 andthree animals found expired on Day 8 of the study in the group alsoreceiving RSLAIL-2 and Anti-PD-1.

TABLE 8 Phase I Summary of Results (Mean ± SE) Mean Mean Tumor RelativeTumor Volume on Tumor Growth Treatment Volume on Inhibition¹ InitiationDay 11 On Day 11 Treatment Dose N (mm³) (%) (%) p value² Vehicle ControlNA 10 177 ± 10 553 ± 49 NA NA (Day 0, 4, 8) RSLAIL-2 (Day 4) + 0.8 mg/kg8 198 ± 16 128 ± 24 76.9 <0.05 Anti CTLA-4 (Day 100 μg 0, 4, 8) RSLAIL-2(Day 4) + 0.8 mg/kg 8 195 ± 14 520 ± 46 5.98 NS³ Anti CTLA-4 (Day 100 μg0, 4, 8) + 100 μg anti-CD8a (Day −2, 0, 7) RSLAIL-2 (Day 4) + 0.8 mg/kg8 177 ± 13 483 ± 51 12.7 NS Anti CTLA-4 (Day 100 μg 0, 4, 8) + 50 μlAnti Asialo-GM1 (Day −2, 0, 7) RSLAIL-2 (Day 4) + 0.8 mg/kg 8 184 ± 10285 ± 46 48.5 <0.05 Anti PD-1 (Day 0, 4, 8) 100 μg RSLAIL-2 (Day 4) +0.8 mg/kg 8 176 ± 10 539 ± 45 2.53 NS Anti PD-1 (Day 0, 4, 8) + 100 μganti-CD8a (Day −2, 0, 7) 100 μg RSLAIL-2 (Day 4) + 0.8 mg/kg Anti PD-1(Day 0, 4, 8) + 100 μg 8 223 ± 15 364 ± 65 34.2 NS Anti Asialo-GM1 50 μl(Day −2, 0, 7) ¹Mean tumor growth inhibition (% TGI) was assessed on Day18 (last day control animals were present) by using the formula: % TGI =(1 − (Relative Tumor Volume (%)^(Treatment Group) ÷ Relative TumorVolume (%)^(Control Group)) × 100 ²One-Way ANOVA with multiplecomparisons, Tukey's post-test. ³NS, did not achieve statisticalsignificance.

This study demonstrates the efficacy of combining immune therapy usingRSLAIL-2 and Anti-CTLA-4 or RSLAIL-2 and Anti-PD-1 in EMT6 murinemammary carcinoma tumors. In addition, the loss of antitumor efficacyfollowing in vivo depletion of NK and CD8 T cells suggests a role forboth cell types in this efficacy.

1. A method of administering to a patient suffering from a cancer, themethod comprising the steps of: (a) an IL-2Rβ-activating amount of along acting, IL-2Rβ-selective agonist; and (b) a CTLA-4pathway-inhibiting amount of an anti-CTLA-4 antibody or a PD-1pathway-inhibiting amount of an anti-PD-1 antibody.
 2. The method ofclaim 1, wherein the patient is human.
 3. The method of any one ofclaims 1 and 2, wherein the cancer is ,a solid cancer.
 4. The method ofany one of claims 1 and 2, wherein the solid cancer is selected from thegroup consisting of breast cancer, ovarian cancer, colon cancer,colorectal cancer, gastric cancer, malignant melanoma, liver cancer,small cell lung cancer, non-small cell lung cancer, thyroid cancers,kidney cancer, cancer of the bile duct, brain cancer, cervical cancer,maxillary sinus cancer, bladder cancer, esophageal cancer, Hodgkin'sdisease and adrenocortical cancer.
 5. The method of claim 3, wherein thecancer is colon cancer.
 6. The method of claim 3, wherein the cancer isbreast cancer.
 7. The method of any one of claims 1 and 2, wherein step(a) is carried out prior to step (b) being carried out.
 8. The method ofany one of claims 1 and 2, wherein (a) is carried out after step (b) iscarried out.
 9. The method of any one of claims 1 and 2, wherein steps(a) and (b) are carried out simultaneously.